Multiple forms of ischemia/reperfusion (IR)-induced tissue injury and inflammation are initiated by neoantigen exposure prior to antibody (Ab) recognition. In mesenteric IR, multiple neoantigens including annexin IV, 2-GPI and non-muscle myosin have been identified by naturally occurring antibodies (NAb) recognition. Each NAb induces tissue injury in the IR- resistant Rag-1-/- mouse but surprisingly peptides derived from each neoantigen also attenuate IR-induced intestinal damage in wildtype mice. This anomaly suggests that the neoantigens may form a complex induced in a sequential manner on the same or distinct cell types. The mechanisms by which the distinct neoantigens interact such that distinct peptides attenuate injury while individual NAb induce injury remains unclear. The proposed research is expected to determine the interactions of the exposed neoantigens that are involved in IR-induced injury and explain the conundrum. Based on our preliminary data and the literature, we will test the hypothesis that TLR4 ligation induces neoantigen expression in a sequential manner forming a complex which may be recognized by any one NAb but also inhibited by distinct peptides. We will test the hypothesis with the following two specific aims: 1 Determine the ability of 2-GPI to initiate a complex of IR-induced neoantigens and 2) determine the role of TLR4 in neoantigen in response to IR. We will test aim 1 by evaluating novel 2-GPI-derived peptides in both in vivo IR studies and in vitro biochemical hypoxia studies. Aim 2 will be tested using chimeric mice and myocardial ischemia/reperfusion in vivo studies which will be complemented with biochemical in vitro studies. The innovative hypothesis that the neoantigens form a complex involving TLR4 explains the requirement for both NAb induction and peptide inhibition of local and systemic tissue damage in response to IR. This contribution is significant as it will resolve the mystery, enhance our knowledge and allow for development of rational therapeutics for multiple IR events ranging from intestinal IR and myocardial infarction to stroke and organ transplants. Understanding the mechanism by which the distinct peptides protect will provide critical targets for prevention or treatment of IR-inducd injury.